According to an technical committee of FAO, “cheese” is defined as a fresh product or a matured product obtained by coagulating milk, cream, skimmed milk or partially skimmed milk, butter milk, or a partial or total combination of these products, followed by discharging whey (“Dairy product manufacture I,” edited by the Editing Committee for Dairy Technology Series Books, 1st ed.; Oct. 30, 1963, Asakura Syoten, Tokyo).
Cheese is roughly classified into natural cheese and processed cheese. The former refers to a fresh product, which has been produced by adding a lactic acid bacterium or a milk-clotting enzyme for curdling, or the matured products thereof. On the other hand, the latter refers to natural cheese which has been processed in such manners wherein the natural cheese is heat-melted and emulsified. Natural cheese is further classified into that produced via a maturing step, such as super hard cheese, hard cheese, semi hard cheese, soft cheese, or the like, and fresh cheese produced without maturing step.
In cheese manufacture, a characteristic step is a curdling step achieved with a milk-clotting enzyme. Naturally, in consideration of the above-described definition of cheese, cheese can be produced by coagulating milk without a milk-clotting enzyme. However, insofar as the present invention is concerned, cheese refers to the one produced by using a milk-clotting enzyme for curdling.
The milk-clotting enzyme is, as known well, called rennet or chymosin which is obtainable by extracting from the abomasum of a calf. In addition, there is rennet derived from other origins such as microorganisms.
A curdling reaction by a milk-clotting enzyme is based on a very fine and sophisticated principle. Milk to be subjected to the curdling reaction (raw material milk) includes bovine milk, goat milk, buffalo milk, reindeer milk, donkey milk, camel milk, and the like. However, not only these whole milk, but also partially skimmed milk, skimmed milk, or powder milks prepared by drying can be used. In each milk, the main component of the protein constituting raw material milk is casein. The reaction of casein caused by a milk-clotting enzyme is an important step for curdling.
Casein is roughly classified into α-, β-, and κ-caseins and, in milk, α- and β-caseins are localized inside and κ-casein is localized outside to make a casein micelle structure through calcium. That is, κ-casein is exposed outside the casein micelle. κ-Casein is a protein containing a sugar and having a molecular weight of about 19000, and has the hydrophilic portion and the hydrophobic portion. The hydrophobic portion is located inside and the hydrophilic portion is outside, and therefore, the casein micelle exists stable in milk.
The milk-clotting enzyme is a protease having a very high substrate specificity, and cleaves the bond between phenylalanine which is the 105th amino acid and methionine which is the 106th amino acid, both from the N terminal of κ-casein. This cleaved point is the boundary point between the hydrophilic portion and the hydrophobic portion. Hence, by the enzymatic reaction of the milk-clotting enzyme, the hydrophilic portion is separated from the κ-casein, and the hydrophobic portion is exposed outside the casein micelle. Individual hydrophobic portions aggregate gradually one another by their interaction to become more instable by the presence of calcium ions, and are precipitated when the temperature is raised. This precipitation is curd, and the water-soluble portion which has not been curdled is separated as whey. The whey fraction contains α-lactoalbumin, β-lactoglobulin, lactose and the like as main components. The curdled fraction is casein and can yield the so-called cheese curd. Cheese curd is subsequently subjected to a salting process to produce natural cheese.
As described above, cheese is yielded by precipitating casein fractions from raw material milk, and therefore, increasing yields thereof is a very important subject from the industrial point of view. Of course, yielding cheese curd in further larger amounts from a certain amount of raw material milk provides higher benefits in various points, including reduction of manufacturing cost, an effective use of milk resources, and providing lower priced products for consumers.
For these reasons, considerable numbers of techniques have been developed to increase the yield of cheese curd. Solving the problem of increasing such yield is closely related to the technical problem of how to incorporate into curd the protein and lactose, which are otherwise discharged as whey.
For example, U.S. Pat. No. 4,205,090 discloses a technique for increasing the yield of cheese curd and, in turn, cheese, using highly concentrated milk by concentration through applying ultrafiltration method. JP-T-1982-501810, a Published Japanese Translation of a PCT Application, discloses a method for preparing cheese by using raw material milk, wherein said raw material milk has been obtained by concentrating selectively a raw material milk by ultrafiltration to increase the ion strength of the raw material milk, followed by fermentation and removal step of the water. In addition, in Japanese Patent Application Laid-open (Kokai) No. 1990-308756 is described that whey yielded as a byproduct upon cheese production is concentrated, and when the resulting concentrated whey protein and a concentrated raw material milk are used to produce cheese, the resulting cheese curd contains the highly concentrated whey protein, which results in the effective use of the whey protein as a byproduct.
However, these techniques require a pretreatment such as ultrafiltration or the like, of raw material milk or whey to be reused, and cannot be said to be convenient industrial methods. In addition, methods of cheese preparation by using raw material milk treated by ultrafiltration causes no effects on the product quality of cheese of a short-term maturation type. However, in the case of cheese of a long-term maturation type, decomposition of the protein and flavor generation of cheese may be inhibited. These defects may be attributable to the facts that, in the case of cheese rich in non-denatured whey protein, the whey protein itself is difficult to decompose, and that the whey protein inhibits decomposition of the casein by a protease (Jameson and Lelierve; Bull. of the IDF, 313: 3-8 (1996), deKoning et al.; Netherlands Milk Dairy J. 35: 35-46 (1981), and Bech; Int. Dairy J. 3: 329-342 (1993)).
In conclusion, the current cheese preparation technique comprising a step of concentrating raw material milk cannot be said to satisfy sufficiently such quality as required by consumers.
On the other hand, in recent years, a cheese preparation technique using a protein-crosslinking enzyme has been reported. The protein-crosslinking enzyme refers herein to a TG. For example, Japanese Patent Application Laid open (Kokai) No. 1989-27471 discloses aproduction example of cheese by using a TG. However, in this case, curdling of milk is carried out not with the milk-clotting enzyme, but by acidification with gluconodeltalactone or lactic acid bacteria and, in addition, it lacks the point “obtainable by discharging whey” described in the definition of cheese made by FAO committee previously mentioned in this description. Therefore, cheese in the case is different from that according to the present invention described in detail later. In addition, in Japanese Patent Application Laid-open (Kokai) No. 1990-131537, a technique of preparing a cheese food by using a TG has been described. The cheese food stated there indicates a processed cheese by using natural cheese as the raw material, and is a food different from natural cheese of the present invention.
WO93/19610 discloses a method wherein a TG is added to an acidic milk protein solution of which the pH has been lowered. However, this method comprises no coagulation step with a milk-clotting enzyme, and therefore, the resultant cheese is different from the natural cheese of the present invention. Also, in WO94/21129 is described a method which comprises using a TG toward an acidic food gel made of a milk protein. However, this method also comprises using no milk-clotting enzyme, and therefore, the resultant cheese is different from the natural cheese of the present invention. WO92/22930 discloses a method of preparation of a milk-like product by using the milk-clotting enzyme, and however, no mention is given of production of cheese itself. In addition, the order in which the milk-clotting enzyme and TG are added to a milk protein solution, which order is one of the essential features of preparation method of cheese curd of the present invention, is never disclosed in this document.
WO94/21130 describes a process for preparing a milk-based non-acidic edible gel, in which a TG is first added to the raw material milk, followed by adding a milk-clotting enzyme, and then the resultant mass is subjected to heating treatment. The heating treatment in this way is a heating treatment at a temperature ranging from 60 to 140° C. after the TG and the milk-clotting enzyme have been added, and this treatment presumably causes the inactivation of the enzyme and formation of the gel. This process comprises no whey separation, which characterizes the inventive cheese preparation, and is remarkably different from the present invention, in the order in which the milk-clotting enzyme and a TG are added and at the temperature range of heating.
On the other hand, WO97/01961 discloses a cheese preparation method in which a TG is added to a raw material milk, followed by adding a milk-clotting enzyme to the resulting mixture, and then the whey is separated. In the description of this disclosure, the TG is reacted at a temperature ranging from 5 to 60° C., preferably from 40 to 55° C. The reaction temperature range of the TG in the cheese curd producing process of the present invention overlaps naturally with that temperature range, because the both temperature ranges are reaction temperature ranges of the same enzyme (TG). According to the present invention, however, it is an essential element to conduct the κ-casein cleaving reaction with a milk-clotting enzyme, prior to the enzyme reaction with a TG. This element characterizes the production process of the present invention, differing evidently from that disclosed in WO97/01961.
Japanese Patent Application Laid-open (Kokai) No. 1996-173032 describes the method in which (1) a TG is added to a raw material milk to carry out the enzyme reaction for a specific time, followed by carrying out a heating treatment to inactivate the TG, and then, a milk-clotting enzyme is added, (2) a milk-clotting enzyme is added to a raw material milk to carry out the reaction for a specific time, and then, a TG is added, or (3) a milk-clotting enzyme and a TG are added to a raw material milk at the same time. Description is made of conducting the enzymatic reaction with a TG at a temperature ranging from 10 to 40° C. in these steps. However, the method obviously differs from that of the present invention wherein a milk-clotting enzyme is allowed to act at a low temperature.
Further, cheese preparation methods by using a TG include the method disclosed in EP1057411, in which the cheese curd is prepared by adding a TG and a protease which is not rennet (non-rennet protease) to a raw material milk. However, this method is characterized by using a non-rennet protease, and therefore, essentially differs from the cheese curd producing method of the present invention. In addition, EP1057412 discloses the cheese preparation method, in which a TG is added to a raw material milk to carry out the reaction for a certain period of time, followed by adding fat, emulsifier, salt and the like, and the resulting mixture is blended with a cheese solution which has already been heat-dissolved separately. However, this method is a preparation method for processed cheese, and differs from the producing method of the present invention for natural cheese. Moreover, in the above-mentioned EP1057411, a method is described, in which whey is added to a raw material milk, whereby a TG is reacted under the condition wherein the concentration of the whey protein is relatively raised, and then curd is obtainable with a milk-clotting enzyme. However, this method is also completely different from that of the present invention.
Use of a TG in preparing curd for natural cheese may include three methods: (1) a TG is directly added to a raw material milk to carry out the reaction for a certain period of time, and then, a milk-clotting enzyme is added to the reaction mass, (2) a milk-clotting enzyme is added to a raw material milk to carry out the reaction for a certain period of time, and then, a TG is added to the reaction mass, and (3) a milk-clotting enzyme and a TG are added to a raw material milk at the same time.
Among these methods, in the method (1), first, a TG reacts on the protein (casein) in the raw material milk, and next, the milk-clotting enzyme works. The milk-clotting enzyme is, as described above, an enzyme having a very high substrate specificity, and thus, may be decreased in its reactivity to the κ-casein modified with a TG (i.e., the enzymatic reaction is inhibited), in other words, the curdling reaction may be inhibited. Lorenzen (Milchwissenschaft 55 (8): 433-437 (2000)) reported a case wherein curdling performance was much decreased for skimmed milk treated actually with a TG.
Next, in the method (2), first, a milk-clotting enzyme is added to a raw material milk. In this case, a TG is absent during the work of the milk-clotting enzyme, and hence, the enzymatic reaction by the milk-clotting enzyme is not inhibited by a TG. However, as the enzymatic reaction by the milk-clotting enzyme proceeds, the K-casein is cleaved to separate a glycomacropeptide being the hydrophilic portion, and simultaneously, the hydrophobicity of the surface of the casein micelle increases. This phenomenon means that the curdling reaction goes at the same time, in other words, milk coagulation occurs. Adding a TG after curdling reaction or curd formation has a problem of difficulty in blending the TG uniformly with curd.
Finally, the method (3) is a method in which a milk-clotting enzyme and a TG are added to a raw material milk at the same time. Also in this case, as in the case of the method (1), there is the problem in which the κ-casein modification occurs as the TG reaction proceeds, and then, the curdling reaction by a milk-clotting enzyme is inhibited.
In consideration of the facts as described above, it is not always preferable to use a TG in the curd preparation in natural cheese production in view of inhibition of the curdling reaction (curd formation being inhibited). Inhibition of the curdling reaction by the TG treatment is actually described in WO92/22930 as described above. However, occurrence of a curdling phenomenon requires a specific temperature (around 30° C.) or higher and relates to the calcium concentration in milk, and therefore, it is not always the fact that the TG treatment does not allow preparing natural cheese. In other words, a raw material milk which has been even subjected to a TG treatment can yield curd by raising the temperature or adding calcium.
The techniques of Japanese Patent Application Laid-open (Kokai) No. 1996-173032 and WO97/01961 as described above are techniques using skillfully those techniques, whereby the effect of a TG to inhibit curdling is avoided, and however, there should be an essential solution elsewhere also. Consequently, the problem is how to avoid curdling inhibition of a TG in cheese curd preparation and how to develop a method for adding the TG effectively.